A cytosolic NAD+ -dependent GPDH from maize (ZmGPDH1) is involved in conferring salt and osmotic stress tolerance

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Plant glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to produce glycerol-3-phosphate (G-3-P), and plays a key role in glycerolipid metabolism as well as stress responses.

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Nội dung Text: A cytosolic NAD+ -dependent GPDH from maize (ZmGPDH1) is involved in conferring salt and osmotic stress tolerance

Zhao et al. BMC Plant Biology (2019) 19:16 https://doi.org/10.1186/s12870-018-1597-6 RESEARCH ARTICLE Open Access A cytosolic NAD+-dependent GPDH from maize (ZmGPDH1) is involved in conferring salt and osmotic stress tolerance Ying Zhao1†, Meng Liu1†, Lin He1, Xin Li2, Feng Wang1, Bowei Yan1, Jinpeng Wei1, Changjiang Zhao1, Zuotong Li1* and Jingyu Xu1* Abstract Background: Plant glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to produce glycerol-3-phosphate (G-3-P), and plays a key role in glycerolipid metabolism as well as stress responses. Results: In this study, we report the cloning, enzymatic and physiological characterization of a cytosolic NAD+- dependent GPDH from maize. The prokaryotic expression of ZmGPDH1 in E.coli showed that the enzyme encoded by ZmGPDH1 was capable of catalyzing the reduction of DHAP in the presence of NADH. The functional complementation analysis revealed that ZmGPDH1 was able to restore the production of glycerol-3-phosphate and glycerol in AtGPDHc-deficient mutants. Furthermore, overexpression of ZmGPDH1 remarkably enhanced the tolerance of Arabidopsis to salinity/osmotic stress by enhancing the glycerol production, the antioxidant enzymes activities (SOD, CAT, APX) and by maintaining the cellular redox homeostasis (NADH/NAD+, ASA/DHA, GSH/GSSG). ZmGPDH1 OE Arabidopsis plants also exhibited reduced leaf water loss and stomatal aperture under salt and osmotic stresses. Quantitative real-time RT-PCR analyses revealed that overexpression of ZmGPDH1 promoted the transcripts accumulation of genes involved in cellular redox homeostasis and ROS-scavenging system. Conclusions: Together, these data suggested that ZmGPDH1 is involved in conferring salinity and osmotic tolerance in Arabidopsis through modulation of glycerol synthesis, stomatal closure, cellular redox and ROS homeostasis. Keywords: Glycerol-3-phosphate dehydrogenase, Glycerol, Antioxidants, Redox homeostasis, Salt stress, Osmotic stress, Maize (Zea mays L.) Background major pathways. In the first route, G-3-P is generated by It has been shown that glycerol-3-phosphate (G-3-P) NAD+-dependent GPDH (EC 1.1.1.8)-mediated reduc- serves as a significant intermediary metabolite that con- tion of DHAP; while in the second route, G-3-P is pro- nects multiple metabolic pathways, such as gluconeo- duced from glycerol through phosphorylation catalyzed genes, glycolysis and glycerolipid synthesis [1, 2]. Recent by glycerol kinase (EC 2.7.1.30) [4]. evidences proved that G-3-P also plays a crucial role in Multiple forms of GPDH have been identified from adapting to adverse stresses, including salinity, patho- eukaryotes, and most of them are proved to be key regu- genic microbes, freezing and anaerobic stresses [3]. In lators in stress responses [5–7]. There are five GPDH higher plants, G-3-P can be biosynthesized through two isoforms in Arabidopsis, which are associated with dif- ferent subcellular organelles: one mitochondrial * Correspondence: lxg6401999@163.com; xujingyu2003@hotmail.com FAD-dependent GPDH (EC 1.1.99.5), two plastidic † Ying Zhao and Meng Liu contributed equally to this work. NAD+-dependent GPDHs and two cytosolic NAD+-de- 1 Key Lab of Modern Agricultural Cultivation and Crop Germplasm pendent GPDHs [5, 8–10]. Previous studies demon- Improvement of Heilongjiang Province, Daqing Key Lab of Straw Reclamation Technology Research and Development, College of Agriculture, strated that the AtGPDHc2 gene encoded a Heilongjiang Bayi Agricultural University, Daqing 163319, China cytosol-targeted GPDH which is involved in Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhao et al. BMC Plant Biology (2019) 19:16 Page 2 of 17 pathogen-elicited defense responses in Arabidopsis via Arabidopsis to salinity and osmotic stresses, with higher its effects on the provision of G-3-P [5]. Plants deficient glycerol level, lower fluctuation of cellular redox status in plastid-localized GPDH (SFD1/GLY1) exhibited a ser- and stronger ROS antioxidant defense in comparison to ious impairment in plastidal glycerolipids pathway of both atgpdhc2 mutant and WT plants. The results showed Arabidopsis and overexpression of SFD1/GLY1 could in- that ZmGPDH1 was pivotal in strengthening salt and os- crease the plastidic lipid contents as well as the photo- motic stress tolerance by regulating glycerol production, synthetic assimilation rate in transgenic rice plants [11]. redox homeostasis and ROS antioxidant defense. In addition to their pivotal role in lipid metabolism, plants GPDHs also participate in modulating the intra- Results cellular redox status through the mitochondrial G-3-P ZmGPDH1 encodes a cytosol-targeted protein with NAD+- shuttle system [8, 9]. In Arabidopsis thaliana, a mito- dependent GPDH activity chondrial FAD-GPDH (EC 1.1.99.5) encoded by the gene One GPDH gene was originally obtained through AtGPDHm1, along with a cytosolic NAD+-dependent BLAST searching against the maize genome utilizing the GPDH (EC 1.1.1.8) encoded by the gene AtGPDHc1, was reported AtGPDHc2 as query [5], the retrieved gene was capable of forming the mitochondrial G-3-P shuttle [8]. designated as ZmGPDH1. The full length CDS of The operation of G-3-P shuttle is of vital importance to ZmGPDH1 was cloned, which had 458 amino acids and preserve the homeostasis of NADH/NAD+ ratio, which an apparent molecular mass of 51 kDa. The complete is a prerequisite for cells to keep normal metabolic activ- CDS sequence of ZmGPDH1 was submitted to Gene- ities. In previous studies, it has been recognized that the Bank with the following accession number: MH460963. expression of AtGPDHc1 and AtGPDHm1 is dramatic- The sequence alignment revealed that ZmGPDH1 exhib- ally induced under a variety of stress conditions, like ited very high protein sequence identity (77%) to oxygen availability, salinity and dehydration [8, 10]. AtGPDHc2, and both proteins consisted of one AtGPDHc1 knock-out mutants are more sensitive to C-terminal GPD domain (PF07479) that represents abscisic acid (ABA) than wild-type (WT) plants, and DHAP-binding site and one N-terminal NAD-binding do- have failed to stabilize the balance of NADH/NAD+ [8]. main (PF01210), suggesting that ZmGPDH1 encodes an Loss of AtGPDHc1 also affected other metabolic path- NAD+-dependent GPDH (Additional file 1: Figure S1). way involved in redox shuttling, such as mitochondrial To certify its subcellular localization, the coding region malate/OAA shuttle [8]. of ZmGPDH1 was fused to the N-terminal end of GFP re- The characteristics of GPDH genes in relation to salin- porter gene, and the construct was transformed into ity or osmotic tolerance have been described in some wild-type (WT) Arabidopsis. The mesophyll protoplasts of halophilic microalga species [6, 12, 13]. A putative phos- p35S-ZmGPDH1::GFP and p35S::GFP (control) transgenic phoserine phosphatase (PSP) domain has been found in Arabidopsis plants were isolated and monitored (Fig. 1a). GPDH isoforms from Dunaliella salina (DsGPDH2, The free GFP was distributed in cytosol as well as nucleus, G3PDH) and Chlamydomonas reinhardtii (CrGPD2), whereas the ZmGPDH1-GFP was specifically located in which can serve as glycerol-3-phosphatase (EC 3.1.3.2.1) cytosol. Meanwhile, the same result was also found in rice enzyme and directly catalyze the conversion of DHAP to mesophyll protoplasts temporarily expressing the glycerol under high osmotic environment [14–16]. Fur- ZmGPDH1-GFP together with the cytosol-marker (mkate thermore, the transcription of mushroom GPDH gene is protein), which demonstrated that ZmGPDH1 was a greatly stimulated by drought and salinity conditions; cytosol-localized protein (Additional file 2: Figure S2). and overexpression of PsGPD improves the salinity tol- To further study the catalytic characteristics of erance of transgenic rice by increasing the osmotic po- ZmGPDH1, the recombinant protein generated by the E. tential and stomatal conductance [17]. coli Rosetta (DE3) strain expressing plasmid of 6 × His Although the importance of GPDH genes in stress re- tagged ZmGPDH1 was purified with a Ni-NTA column. sponses is well documented in yeast, algae as well as a A ZmGPDH1-His fusion protein with an expected size few plants, there is scarce information about their func- of 65 kDa (consisting of target gene and histidine tions in field crops. Salt and osmotic stresses are the marker) was identified by SDS/PAGE and Western blot major environmental factors that seriously influence (Fig. 1b, c and d). The recombinant ZmGPDH1 protein crop growth and productivity [18]. Here, we isolated and was assayed for its kinetic properties relative to the sub- characterized a cytosol-localized GPDH (ZmGPDH1) strate DHAP. Using Eadie-Hofstee plot, the Km and gene from maize, which had functional NAD+-depen- Vmax of DHAP were estimated as 2.75 mM and 0.071 dent GPDH activity, and apparent transcriptional re- umol·min− 1·mg− 1 protein, respectively (Fig. 1e); besides, sponse to salinity and mannitol treatments. In addition, addition of NADH strongly stimulated the enzyme activ- overexpression of ZmGPDH1 in AtGPDHc-deficient mu- ity (Fig. 1f ). These results indicated that the purified tant and WT lines enhanced tolerance of transgenic ZmGPDH1 protein was able to catalyze the reduction of Zhao et al. BMC Plant Biology (2019) 19:16 Page 3 of 17 Fig. 1 ZmGPDH1 encodes a cytosol-targeted protein with GPDH activities. a Subcellular localization of ZmGPDH1. Confocal microscopy observation of pBI121-ZmGPDH1::GFP or pBI121-GFP in transgenic Arabidopsis mesophyll protoplasts. Bars = 10 μm. b Coomassie-stained 12% SDS-PAGE of cell extract from (Lane 1) Rosetta (DE3) Escherichia coli strain and (Lane 2–5) DE3 expressing His-tagged ZmGPDH1 protein. c Coomassie-stained 12% SDS-PAGE of (Lane 1) purified ZmGPDH1 fusion protein. d Western blot analysis of (Lane 1) purified ZmGPDH1 fusion protein using anti-6 × His antibody as probe. e The kinetic properties of ZmGPDH1 with regard to DHAP. f GPDH enzyme activities from purified ZmGPDH1 fusion protein. The reaction was performed in the presence (+) and absence (−) of NADH and DHAP, respectively DHAP with the assistant of NADH. Furthermore, the 5-week-old proZmGPDH1::GUS transgenic plants was optimum pH of the enzyme activity was determined to used as calibrator (Fig. 2m). Consistent with the result of be pH 7.0 and the optimum temperature was 35 °C, re- GUS activity assay, the GUS transcripts could be ob- spectively (Additional file 3: Figure S3). served in all tissues examined, with high levels of tran- scription in roots, flowers and rosette leaves. These Expression profiles of ZmGPDH1 in response to NaCl or results suggested that the ZmGPDH1 promoter exhib- mannitol treatment ited a tissue-specific expression pattern. To examine the potential functions of ZmGPDH1 in re- It has been proven that GPDH genes are essential for sponse to plant growth, the promoter region of stress adaptations in yeast, marine algae and Arabidopsis ZmGPDH1 was amplified and fused to N-terminus of [5, 17–19]. Therefore, to explore its possible involve- GUS reporter gene, and the proZmGPDH1::GUS con- ment in stress responses in maize, we first analyzed the struct was transformed to WT Arabidopsis plants. In transcripts accumulation of ZmGPDH1 under different 4-day-old transgenic seedlings, high GUS activity was stress treatments. The qRT-PCR results showed that observed in shoots apical meristem; in 7-day-old or ZmGPDH1 was remarkably up-regulated by both salinity 14-day-old seedlings, high GUS activity was observed in and osmotic stresses in maize roots, which reached the young leaves and petioles (Fig. 2). In 5-week-old highest level at 3 h under both treatments (Fig. 2n and proZmGPDH1::GUS transgenic plants, strong constitu- o). Notably, the expression of proZmGPDH1::GUS was tive GUS activity was shown in flowers, roots, rosette also enhanced in the presence of NaCl and mannitol leaves, and stems. To further verify these findings, quan- treatments (Fig. 2p), suggested that ZmGPDH1 was in- titative real-time RT-PCR (qRT-PCR) was carried out, volved in the transcriptional response during salt or os- and the level of GUS transcripts in siliques of motic adaptions. Zhao et al. BMC Plant Biology (2019) 19:16 Page 4 of 17 Fig. 2 Expression characteristics of ZmGPDH1 gene. ZmGPDH1 promoter-GUS assay in various tissues, including (a) rosette leaf, (b) stem, (c) cauline leaf, (d) mature silique, (e) flower, (f) flower bud, (g) stigma, (h) root, (i) 4-day-old plant, (j) 7-day-old plant, (k) 14-day-old plant and (l) immature silique. (m) The transcript level of GUS gene in different tissues of 5-week-old proZmGPDH1::GUS transgenic plants (T3 generation), including stems (ST), rosette leaves (RL), cauline leaves (CL), roots (RT), flowers (FL), flower buds (FLB), siliques (SL). The expression of GUS gene in SL was used as a calibrator. The transcriptional response of ZmGPDH1 in maize roots exposed to (n) NaCl or (o) mannitol treatments. The expression of ZmGPDH1 in untreated samples (control) harvested at each time point was used as a calibrator. (p) The analysis of GUS activity in response to mannitol or NaCl treatment. 7-day-old proZmGPDH1::GUS transgenic seedlings were transferred to either half-strength MS plates (1/2 MS), plates with 300 mM mannitol or plates with 150 mM NaCl for 12 h before GUS staining. Non-transformed wild-type (WT) was used as a control. Bars = 100 μm. The asterisks represented a significant difference as determined by the Student′s t-test (*P < 0.05, ** P < 0.01) Overexpression of ZmGPDH1 enhanced the tolerance of lines (Fig. 3a). The enzymatic assay revealed that GPDH Arabidopsis to salt and osmotic stresses activities of ZmGPDH1 overexpression lines (OE-1, OE-2) Next, to further understand how ZmGPDH1 responds to were 2.1- to 2.3-fold higher than that in the WT, while salt or osmotic stress, ZmGPDH1 transformed GPDH activities of atgpdhc2 mutant was 31% of that in AtGPDHc2-deficient mutant (COM-1, COM-2) and WT the WT. Meanwhile, the GPDH activities of ZmGPDH1 (OE-1, OE-2) lines were generated. The T-DNA insertion complementation lines (COM-1, COM-2) were 1.2- to mutant of AtGPDHc2 was identified by PCR and 1.3-fold higher than that in the WT, which indicated that reverse-transcription PCR (RT-PCR), and the homozygous ZmGPDH1 was successfully expressed and functioned atgpdhc2 mutants were selected for the transformation with GPDH activity in both OE and COM lines (Fig. 3b). studies (Additional file 4: Figure S4). The RT-PCR results Compared with WT, atgpdhc2, COM and OE lines showed that the full-length transcription product of showed no aberrant phenotype under normal growth con- ZmGPDH1 was absent in the WT Arabidopsis or ditions, no matter in the vegetative growth phase or repro- atgpdhc2 mutant, but existed in all ZmGPDH1 transgenic ductive developmental stages (Fig. 3c). Zhao et al. BMC Plant Biology (2019) 19:16 Page 5 of 17 Fig. 3 Germination characteristics of the ZmGPDH1 transgenic lines in response to salt or osmotic stresses. a Reverse transcription PCR (RT-PCR) of ZmGPDH1 transcripts in the WT, atgpdhc2, ZmGPDH1 transformed atgpdhc2 (COM-1, COM-2) and transgenic WT (OE-1, OE-2) plants. ACTIN served as the internal reference. b The GPDH activities in ZmGPDH1 transgenic lines compared with the WT and atgpdhc2. c Morphological comparison among the WT, atgpdhc2, COM-1, COM-2, OE-1 and OE-2 lines grown in soil. Top panel, 28-day-old plants; bottom panel, 56-day-old plants. Bars = 200 mm. d The germination rate of the six lines under normal (control), NaCl or mannitol conditions at day 5 after imbibition. e Images of seeds germinated on either half-strength MS medium (control), medium with 100 mM NaCl or medium with 200 mM mannitol for 7 days. Bars = 50 mm. Asterisks indicated significant differences from the WT, as determined by Student′s t-test (*P < 0.05, ** P < 0.01) Additionally, to evaluate the performance of transplanted to half-strength MS plates supplemented ZmGPDH1 transgenic lines in response to salt or os- with 300 mM mannitol or 150 mM NaCl for 7 days. As motic stresses, the seeds of WT, atgpdhc2, COM and shown in Fig. 4a, the atgpdhc2 seedlings displayed much OE lines were germinated on half-strength MS mediums severer stress-hypersensitive phenotype than the WT, containing 100 mM NaCl or 200 mM mannitol. As with a partial leaf bleaching phenomenon. However, shown in Fig. 3d and e, seed germination of atgpdhc2 ZmGPDH1 transformed atgpdhc2 (COM-1, COM-2) was severely delayed in comparison to the WT, whereas plants showed wild-type-like phenotype under NaCl and the germination rate of ZmGPDH1 OE seeds was much mannitol treatments, indicating that overexpression of higher than that of other lines. The ZmGPDH1 COM ZmGPDH1 could rescue salt and osmotic sensitivity of seeds displayed stress-sensitive morphologies similarity atgpdhc2 mutants. In addition, ZmGPDH1 OE plants ex- to the WT, albeit they had a relative higher germination hibited an enhanced tolerance to salt/osmotic stress, and rate (Fig. 3d and e). a resulting higher fresh weight and root length com- To investigate the impact of ZmGPDH1 overexpres- pared with the WT (Fig. 4 b and c). sion on salt/osmotic resistance at early seedling stage, Likewise, when the 3-week-old Arabidopsis plants 7-day-old WT, atgpdhc2, COM and OE lines were were subjected to 400 mM mannitol or 200 mM NaCl Zhao et al. BMC Plant Biology (2019) 19:16 Page 6 of 17 Fig. 4 Overexpression of ZmGPDH1 enhances the salt- and drought-tolerance in transgenic Arabidopsis. a Images of 7-day-old WT, atgpdhc2, complementation (COM-1, COM-2) and overexpression (OE-1, OE-2) plants grown on either half-strength MS plates (control), plates with 300 mM mannitol or 150 mM NaCl for 7 days. Bars = 100 mm. b and c The primary root length and fresh weight of the six lines after 7 days of treatments. Images of 3-week-old plants irrigated with water (control), 200 mM NaCl (d) or 400 mM mannitol (e) every 3 days for 9 days. Bars = 200 mm. The total chlorophyll contents and chlorophyll fluorescence (Fv/Fm) of 3-week-old plants irrigated with water (control), 200 mM NaCl (f) or 400 mM mannitol (g) over 9 days. Asterisks indicated significant differences (*P < 0.05, ** P < 0.01) from the WT, as determined by Student′s t-test treatment for 9 days, the growth of atgpdhc2 mutants ZmGPDH1 regulates glycerol-3-phosphate and glycerol was strongly inhibited compared with the WT (Fig. 4d levels under salt and osmotic stresses and e). Conversely, the ZmGPDH1 OE or COM plants Glycerol is an important compatible solute and the showed obviously improved tolerance to salinity or os- physiological significance of glycerol biosynthesis under motic stress relative to the other lines (Fig. 4d and e). salinity or osmotic condition has been reported in many Synchronously, the chlorophyll fluorescence parameter species [4, 12, 20]. G-3-P is a primary substrate for gly- (Fv/Fm) and total chlorophyll content were analyzed in cerol synthesis [21]. To characterize the effect of atgpdhc2, COM-1, WT and OE-1 plants. Under stand- ZmGPDH1 on glycerol metabolism, we assayed G-3-P ard conditions, chlorophyll content and Fv/Fm had no and glycerol levels in 3-week-old WT, atgpdhc2, COM differences among all four lines (Day 0); however, after and OE Arabidopsis lines treated with 200 mM NaCl or exposure to 400 mM mannitol or 200 mM NaCl treat- 400 mM mannitol for 6 days. Under normal soil condi- ment, atgpdhc2 mutant demonstrated a remarkable tions, significant reduction in G-3-P and glycerol con- reduction in Fv/Fm and chlorophyll content, whereas tents were observed in gpdhc2 mutants, while the the Fv/Fm ratio and total chlorophyll content in OE-1 average contents of G-3-P and glycerol were increased and COM-1 were much higher than that in WT plants in the OE and COM lines compared with WT Arabidop- (Fig. 4 f and g). These data suggested that overexpres- sis (Fig. 5 a and b). After treatment with NaCl or manni- sion of ZmGPDH1 helped to enhance the photochem- tol, the OE and COM plants accumulated higher level of ical efficiency in transgenic Arabidopsis. G-3-P and glycerol than the other lines (Fig. 5a and b), Zhao et al. BMC Plant Biology (2019) 19:16 Page 7 of 17 Fig. 5 Overexpression of ZmGPDH1 increases the glycerol-3-phosphate and glycerol levels under salt and osmotic stresses. a and b The glycerol-3- phosphate and glycerol contents in 3-week-old WT, atgpdhc2, COM-1, COM-2, OE-1 and OE-2 seedlings irrigated with water (control), 400 mM mannitol and 200 mM NaCl every 3 days for 6 days. Asterisks indicated significant differences from the WT by Student′s t-test (*P < 0.05, ** P < 0.01) indicating that the overexpression of ZmGPDH1 could change among the six lines under normal growing con- promote the cellular glycerol biosynthesis under high ditions (Fig. 6b and c). However, when the plants were salinity or hyperosmotic stress. treated with NaCl or mannitol, the ZmGPDH1 OE lines maintained relative higher ASA and GSH contents com- ZmGPDH1 is essential for redox homeostasis under salt pared with the WT, leading to an even higher ASA/ and osmotic stresses DHA or GSH/GSSG ratio. By contrast, there was a no- It has been reported that the cytosolic NAD+-GPDH is in- ticeable decline in reduced ascorbate or glutathione as volved in NADH:NAD+ recycling by catalyzing the con- well as the redox ratio (ASA/DHA, GSH/GSSG) in version of DHAP to G-3-P using NADH as a reducing atgpdhc2 mutants relative to WT or COM lines (Fig. 6b equivalent [8]. To validate if overexpression of ZmGPDH1 and c). Collectively, these results implied that the founc- could affect NADH/NAD+ homeostasis upon salt and os- tion of ZmGPDH1 in salinity and osmotic tolerance motic stresses, the fluctuation in redox status of NADH could partly attribute to sustaining the cellular redox was monitored. Under normal growth condition, no sig- homeostasis. nificant differences were observed in NADH, NAD+ con- tents and NADH/NAD+ ratio among WT, atgpdhc2, ZmGPDH1 regulates the ROS level and cell death under COM-1, COM-2, OE-1 and OE-2 lines (Fig. 6a). However, salt and osmotic stresses a severe interference in NADH/NAD+ homeostasis ap- In plant stress reactions, the redox state is highly corre- peared in all the six lines under salt or mannitol treat- lated with the cellular ROS producing and processing ment, differences could also be seen in the individual [22, 23]. Hence, the remarkable changes in redox ratios NADH or NAD+ contents. Although stress treatments el- (NADH/NAD+, ASA/DHA, GSH/GSSG) in ZmGPDH1 evated the NADH level in each line, the ZmGPDH1 OE transformed plants promoted us to investigate the ROS plants accumulated comparatively lower NADH and level under salt and osmotic stresses. The Fig. 7a higher NAD+ contents in comparison to WT plants, depicted NBT staining of O2.-, while the Fig. 7b illus- resulting in a decreased NADH/NAD+ ratio (Fig. 6a). trated DAB staining of H2O2. In both cases, heavier col- Nevertheless, atgpdhc2 mutants accumulated more oration, reflecting the elevated level of ROS, was NADH and substantially less NAD+ content than WT or detected in atgpdhc2 mutants after NaCl or mannitol COM plants, leading to a higher NADH/NAD+ ratio. treatment. On the contrary, the slighter coloration of These results illustrated that overexpression of ZmGPDH1 NBT and DAB staining were observed in both COM and might facilitate the oxidation of the excessive reductant OE plants under identical conditions. Meanwhile, quan- (NADH) induced by salt and osmotic stresses, thus in- titative measurements showed that the contents of H2O2 creased the NAD+/NADH ratio. and O2.- in COM and OE plants were markedly lower To further examine whether overexpression of than that in the WT and atgpdhc2 mutants, suggesting ZmGPDH1 could influence the other redox couples, the the vital role of ZmGPDH1 in modulating the cellular cellular oxidized and reduced pools of ASA and GSH ROS accumulation under salt or osmotic stress (Fig. 7c were also determined. Similarly, the contents of ASA, and d). The increased ROS production had the potential GSH and their oxidized form DHA, GSSG did not to trigger the compensatory responses of antioxidant Zhao et al. BMC Plant Biology (2019) 19:16 Page 8 of 17 Fig. 6 Overexpression of ZmGPDH1 maintains the redox homeostasis under salt and osmotic stresses. a NADH contents, NAD+ contents, NADH/NAD+ ratios; b ASA contents, DHA contents, ASA/DHA ratios; c GSH contents, GSSG contents, GSH/GSSG ratio in 3-week-old WT, atgpdhc2, COM-1, COM-2, OE-1 and OE-2 seedlings irrigated with water (control), 400 mM mannitol and 200 mM NaCl every 3 days for 6 days. Asterisks indicated significant differences from the WT by Student′s t-test (*P < 0.05, ** P < 0.01) enzymes, therefore, the activities of ROS-scavenging en- overexpression of ZmGPDH1 alleviated the cellular ROS zymes, including catalase (CAT), ascorbate peroxidase accumulation by improving antioxidant defense and (APX) and superoxide dismutase (SOD) were also deter- consequently minimize the cell death as well as mem- mined in this study [23]. As expected, an enhanced ac- brane lipid peroxidation under salt and osmotic stresses. tivity of these antioxidant enzymes was detected in all the lines under salinity or osmotic condition, while the elevation in OE or COM lines was more prominent than ZmGPDH1 is involved in stress-induced stomatal closure that in the WT (Fig. 7e, f and g). In reverse, the The water loss measurement revealed that overexpres- atgpdhc2 mutant possessed relatively lower antioxidant sion of ZmGPDH1 remarkably enhanced the transgenic enzymes activities compared with the WT. plants resistance to water deficit, as reflected by a lower Additionally, the oxidative stress mediated cell death WLR (Fig. 9a). Since water loss mainly depends on the was also detected by Evan′s blue and propidium iodide stomatal regulation, we further valuated whether (PI) staining [24]. As shown in Fig. 8a and b, mild inten- ZmGPDH1 was involved in the modulation of the sto- sities of Evan′s Blue and PI staining was observed in all matal closure under stress treatments. There was no sig- the six lines under the normal conditions. However, the nificant difference in guard cells size or stomatal cell death was strongly stimulated in atgpdhc2 mutants, aperture ratio among WT, atgpahc2, COM-1 and OE-1 moderately stimulated in WT or COM and mildly stim- lines prior to stress treatment (Fig. 9b). By contrast, the ulated in OE line after treatment with NaCl or mannitol NaCl or mannitol treatment markedly induced the sto- (Fig. 8a and b). The TBARS and electrolyte leakage matal closure in all the lines; nevertheless, the stomatal levels, as the measures of oxidative damages in cell aperture ratio was dramatically lower in OE-1 plants and membranes, were significantly lower in OE line than that higher in atgpdhc2 mutants, compared with WT plants. in the other lines under salt and osmotic stresses (Fig. 8c In addition, the stomatal aperture in COM-1 plants was and d). Clearly, these findings indicated that generally similar with that in the WT (Fig. 9c). This Zhao et al. BMC Plant Biology (2019) 19:16 Page 9 of 17 Fig. 7 Overexpression of ZmGPDH1 enhances the ROS scavenging capacity under salt and osmotic stresses. Photographs showing representative (a) NBT and (b) 3,3-diaminobenzidine (DAB) staining of 3-week-old WT, atgpdhc2, COM-1, COM-2, OE-1 and OE-2 plants treated with water (control), 200 mM NaCl and 400 mM mannitol for 2 h. Bars = 50 mm. O2.- (c) and H2O2 (d) levels in the six lines after 6 days of water (control), 200 mM NaCl or 400 mM mannitol treatment. Enzyme activity of (e) superoxide dismutase (SOD), (f) catalase (CAT) and (g) ascorbate peroxidase (APX) in the six lines treated as above. Asterisks indicated significant differences from the WT by Student′s t-test (*P < 0.05, ** P < 0.01) implied that ZmGPDH1 played an essential role in regu- of a number of key genes involved in (1) ASA and GSH lating the stomatal response to salt/osmotic stress. metabolism: cytosolic monodehydro-ascorbate reductase The phytohormone ABA has the ability to induce sto- (MDAR3), cytosolic glutathione reductase (GR1), cyto- matal closure [25]; however, overexpression of ZmGPDH1 solic dehydroascorbate reductase (DHAR1, DHAR2), led to ABA insensitivity in stomatal movement (Fig. 9b). cytosolic glutathione transferase (GSTF14) and cytosolic In the presence of ABA, the stomatal closure was signifi- L-galactose dehydrogenase (GalLDH) [26–28] (2) cantly triggered in atgpdhc2 mutants, moderately trig- ROS-scavenging system: cytosolic copper/zinc super- gered in WT or COM and mildly triggered in OE plants, oxide dismutase (CSD1), cytosolic catalase (CAT1) and illustrating that ZmGPDH1-mediated stomatal closure cytoplasmic ascorbate peroxidase (APX1) [29, 30]. As was independent of ABA (Fig. 9c). In addition, the H2O2 shown in Fig. 10, the transcripts of all genes tested in production in the guard cells was also determined by atgpdhc2 and OE-1 lines were generally similar to WT H2DCF-DA staining. As shown in Fig. 9b, the H2O2 accu- under normal conditions. Upon salinity or osmotic treat- mulation was less in OE plants and more in atgpdhc2 mu- ment, the transcripts of genes participating in ASA-GSH tant compared with that in the WT, suggesting that redox cycle (MDAR3, GR1, DHAR1, DHAR2) were de- overexpression of ZmGPDH1 contributed to sustain the creased in atgpdhc2 mutant but elevated in ZmGPDH1 ROS levels during stomatal movements. OE lines compared with the WT (Fig. 10a). Similarly, the transcripts of genes related to the biosynthesis of Effects of ZmGPDH1 on expression of genes involved in ASA and GSH (GSTF14, GalLDH) were also signifi- redox homeostasis and ROS-scavenging system cantly increased in OE plants, despite which is not the Next, to elucidate the impact of ZmGPDH1 on molecu- case in atgpdhc2 mutants. In addition, the transcripts of lar basis of salinity or osmotic stress response of cellular CSD1, CAT1 and APX1 were highly stimulated by NaCl redox and ROS homeostasis, we detected the transcripts or mannitol treatment, and were higher in ZmGPDH1 Zhao et al. BMC Plant Biology (2019) 19:16 Page 10 of 17 Fig. 8 Overexpression of ZmGPDH1 alleviates the stress-induced membrane injury and cell death. a The Evan′s Blue staining in 3-week-old WT, atgpdhc2, COM-1, COM-2, OE-1 and OE-2 lines irrigated with water (control), 400 mM mannitol and 200 mM NaCl for 2 h. Bars = 50 mm. b The propidium iodide (PI) fluorescence staining in root tips of 7-day-old plants treated on either half-strength MS plates (control), plates with 300 mM mannitol or 150 mM NaCl for 12 h. The images were obtained by cofocal microscope. Bars = 100 μm. TBARS contents (c) and the relative electrolyte leakage (d) of the six lines after 6 days of water (control), 200 mM NaCl or 400 mM mannitol treatments. Asterisks indicated significant differences from the WT by Student′s t-test (*P < 0.05, ** P < 0.01) OE line but lower in the atgpdhc2 compare to the WT, in- protein domains (PF07479, PF01210). The conserved dicating that overexpression of ZmGPDH1 up-regulated GAGAWG motif was found at residues 44–50 of the pro- the expression of the cytosolic antioxidant-related genes tein sequence of ZmGPDH1 (Additional file 1: Figure S1), under both salt and osmotic stresses (Fig. 10b). These data which was similar to the previously reported NAD+-depen- also supported the finding that antioxidant activities of dent GPDH isoforms with an analogous NAD+-binding APX, SOD and CAT increased in the ZmGPDH1 OE fragments corresponding to GXGXXG [8, 10, 32]. transgenic plants. Taken together, these results indicated Enzymatic assay of recombinant ZmGPDH1 proteins that ZmGPDH1 might function in the regulation of cellu- expressed in Escherichia coli Rosetta strain (DE3) (Fig. 1e lar redox and ROS homeostasis to prevent the oxidative and f) showed the purified ZmGPDH1 protein had sub- damage caused by salt or osmotic stress. strate affinity (KmDHAP of 2.75 mM) (Fig. 1e), which com- pared well with the kinetic parameters previously reported Discussion for other GPDH enzymes [10, 33, 34]. The stable or tran- Maize (Zea mays L.) is an important cereal crops as well as sient expression of a green fluorescent protein a major source of biofuel, industrial material and animal (GFP)-tagged ZmGPDH1 in Arabidopsis or wild-type rice feed [31]. Although a great deal of research has indicated were conducted, and both evidenced that ZmGPDH1 pro- that glycerol-3-phosphate dehydrogenase (GPDH) plays a teins were specially targeted to cytosol (Additional file 2: pivotal role in plant growth and stress adaptions [3, 17, Figure S2 and Fig. 1a). The earlier identified Arabidopsis 21], little is currently known about its functions in field GPDH proteins (AtGPDHc1 and AtGPDHc2) were pre- crops including maize. In this study, we isolated a GPDH dicted to be cytosol-located GPDs owing to the absence of gene encoding NAD+-dependent GPDH from maize. Simi- apparent transmembrane regions and subcellular targeting lar to other typical NAD+-dependent GPDH [14, 16, 19], sequences, however, a clear experimental evidence was ZmGPDH1 protein contained the necessary and specific lacking [5, 8]. Zhao et al. BMC Plant Biology (2019) 19:16 Page 11 of 17 Fig. 9 Overexpression of ZmGPDH1 promotes the stomatal closure under salt and osmotic stresses. a The analysis of water loss rate (WLR) of the WT, atgpdhc2, COM-1 and OE-1. b For stomatal closure assays, the abaxial epidermis of rosette leaves were incubated in the light for 1 h to induce the stomatal opening and then treated with water (control), 300 mM mannitol, 150 mM NaCl and 20 μM ABA for 3 h. Bars = 25 μm. c Stomatal aperture was investigated by measuring the length and width of guard cells. Asterisks indicated significant differences from the WT, as determined by Student′s t-test (*P < 0.05, ** P < 0.01) In succession, the physiological functions of the cyto- in other species: overexpression of an oyster mushroom solic ZmGPDH1 gene in mediating salinity/osmotic adap- GPDH gene (PsGPD) increased the salt tolerance in trans- tion were investigated in this study. The transcript genic potatoes and rice; and overexpression of a black abundance of ZmGPDH1 was markedly increased under yeast GPDH gene (HwGPD1B) also enhanced NaCl toler- NaCl and mannitol treatments (Fig. 2n and o). On the ance of Saccharomyces cerevisiae gpd1 mutant [17, 35]. other hand, the transgenic Arabidopsis harboring the We also found that ZmGPDH1 gene was required ZmGPDH1 promoter fused to a GUS reporter gene also for glycerol generation. Glycerol is an important showed relatively higher GUS activity under NaCl or man- osmo-protectant and its accumulation can compen- nitol condition (Fig. 2p), indicating that ZmGPDH1 gene sate for differences between intracellular and extra- was regulated at the transcription level in response to salt cellular water potentials under hyperosmotic environment or osmotic stress, which was consistent with the expres- [6, 12, 13]. In our study, overexpression of ZmGPDH1 sion patterns of other GPDH genes from A.thaliana markedly increased the levels of G-3-P and glycerol, (AtGPDHc1, AtGPDHm1), C. reinhardtii (CrGPDH2, demonstrated that ZmGPDH1 played essential roles in CrGPDH3), D. salina (DsGPDH2, G3PDH) and D. viridis plant adaptation to hypersaline or hyperosmotic shock (DvGPDH1, DvGPDH2) [6, 8, 9, 13, 19]. Furthermore, by contributing to the glycerol biosynthesis (Fig. 5). overexpression of ZmGPDH1 strongly enhanced the toler- Likewise, of the five GPDH enzymes in Chlamydomo- ance of Arabidopsis (WT) to salt/osmotic stress and res- nas reinhardtii, CrGPDH2 and CrGPDH3 were shown cued the salt/osmotic sensitivity of atgpdhc2 mutant, as to be necessary for osmotic-induced glycerol produc- reflected by a pronounced elevation in germination rate, tion [36]. Also, loss of AtGPDHc1 gene encoding a fresh weight, root length, biomass, chlorophyll content cytosol-localized GPDH of Arabidopsis caused the and Fv/Fm ratio under salinity or osmotic conditions hypersensitivity to salt stress due to the severe im- (Figs. 3 and 4). Similar results have been reported earlier pairment in provision of glycerol [8]. Zhao et al. BMC Plant Biology (2019) 19:16 Page 12 of 17 Fig. 10 Overexpression of ZmGPDH1 increases the transcripts of genes involved in cellular redox and ROS homeostasis. a and b Transcripts of genes involved in redox homeostasis (MDAR3, DHAR1, DHAR2, GR1, GSTF14 and GaILDH). c Transcripts of genes involved in ROS-scavenging system (CSD1, CAT1 and APX1). The roots of 3-week-old WT, atgpdhc2, and OE lines were submerged with water (control), 300 mM mannitol or 150 mM NaCl solution for 24 h, respectively. The expression of each gene in the WT treated with water was used to normalize its transcripts in different lines under different conditions. Asterisks indicated significant differences from the WT by Student′s t-test (*P < 0.05; **P < 0.01) Most notably, cytosolic GPDHs are reported to partici- involved in the biosynthesis of ASA and GSH, respect- pate in the mitochondrial G-3-P shuttle system, which ively [28]. In the present study, the increased expression functions as a pivotal route to keep the cellular redox of the marker genes corresponding to the above men- status in Arabidopsis [8]. In this study, ZmGPDH1 over- tioned enzymes in the ZmGPDH1-OE plants implied expression Arabidopsis showed significantly decreased that ZmGPDH1 might exert effects on the ASA/GSH NADH level accompanied by an increased NAD+ accu- redox cycles as well, and play a critical role in optimizing mulation during salt/osmotic stress (Fig. 6a). As a conse- the cellular redox homeostasis of NADH/NAD+, ASA/ quence, a noticeable reduction in cellular NADH/NAD+ DHA and GSH/GSSG under salt and osmotic stresses, ratio was detected in OE lines, suggested that as speculated in Fig. 11. Overexpression of ZmGPDH1 ZmGPDH1 involved in the regulating of NADH/NAD+ also caused a reduction in ROS level, including the redox homeostasis by consuming the excessive redun- guard cell ROS, as exhibited by the slight H2DCF-DA dant NADH and regenerating NAD+ under salt or os- staining under stress treatments (Fig. 7 and Fig. 9). Be- motic stress. In addition, an apparent increase in ASA sides, the level of lipid peroxidation and cell death in and GSH contents as well as their redox pool were ob- ZmGPDH1 OE lines was much lower than that of the served in ZmGPDH1 overexpression Arabidopsis com- other plants (Fig. 8), illustrating that overexpression of pared with that in WT plants (Fig. 6b and c), indicated ZmGPDH1 availably protected cell from oxidative dam- that overexpression of ZmGPDH1 also affected the age and maintained the membrane integrity under salt redox states of ASA and GSH, apart from a decreased or osmotic stress. It has also been reported that the sol- cellular NADH/NAD+ ratio. It is known that GR, uble redox couples seem to assume a dual role with re- MDHAR and DHAR are responsible for the regener- spect to ROS [28]. On one hand, the excessive NADH ation of ASA and GSH in ASA-GSH redox cycle [37]. can be involved in, or related to ROS-generation meta- GalLDH and GST are proved to be essential enzymes bolic pathway by triggering the over-reduction of Zhao et al. BMC Plant Biology (2019) 19:16 Page 13 of 17 Fig. 11 Model of the involvement of ZmGPDH1 in salt/osmotic stress responses. The stress signals triggered the expression of ZmGPDH1 and increased the NAD+-GPDH enzyme activity, which was required for two biological processes: glycerol biosynthesis by affecting the G-3-P provision and NADH/NAD+ homeostasis by disposing of extra reducing power. The elevated expression of genes involved in ROS and redox homeostasis resulted in improved ROS scavenging capacity and a series of positive physiological changes. In addition, the boosted accumulation of G-3-P/glycerol and optimized stomatal movement/water loss were also in concurrence, and collectively contributed to an enhanced salt/ osmotic resistance. Red arrows indicated the tendency of changes molecular oxygen O2 [38]. In contrast, the reductive Additionally, our results showed that ZmGPDH1-OE detoxification of ROS also heavily depends on the plants exhibited greater stomatal closure compared with NADH oxidation, as signified by the increased NAD+/ other lines, indicating that ZmGPDH1 played a key role NADH ratio, which helps to enhance the oxidant scav- in manipulating the stomatal closure under salt or os- enging capacity [23, 28]. Hence, it appeared that motic stress (Fig. 9). However, the ABA-induced stoma- ZmGPDH1 might participate in the regulation of ROS tal closing was impaired in ZmGPDH1-OE lines but metabolism by manipulating the cellular NADH/NAD+ significantly induced in atgpdhc2 mutant, suggesting that ratio (Fig. 11). On the other hand, the antioxidant sys- the ZmGPDH1-mediated stomatal closure might be in- tem has been proved to be dramatically evoked to elim- dependent of ABA. Meanwhile, we found that overex- inate excess ROS under abiotic stress [39]. In pression of ZmGPDH1 reduced the plant sensitivity to agreement with this, we found that the activities and ABA at seed germination and early seedling develop- transcripts of ROS-scavenging enzymes (CSD1, CAT1, mental stages (Additional file 5: Figure S5), which was and APX1) were strongly stimulated by salinity or man- also observed previously on AtGPDHc1 mutants [8]. nitol in OE or COM plants (Fig. 7 and Fig. 10). In sum- Further studies will be needed to interpret the inter- mary, the overexpression of ZmGPDH1 resulted in action of ZmGPDH1 and ABA signaling pathway. higher expression of genes encoding key enzymes of cytosolic ROS scavenging system involving the SOD/ Conclusions CAT/ascorbate/ glutathione cycle, which led to lower We reported the characterization of a cytosolic NAD+-- ROS accumulation and higher ROS detoxification cap- dependent GPDH gene from maize, ZmGPDH1, which acity, and hence stronger stress tolerance (Fig. 11). had profound effects on salt/osmotic tolerance by Zhao et al. BMC Plant Biology (2019) 19:16 Page 14 of 17 regulating the glycerol accumulation, cellular redox according to the reported protocol [41]. The images were homeostasis, ROS-scavenging system as well as stomatal visualized by stereo microscope (Olympus, Japan). movement (Fig. 11). Recombinant ZmGPDH1 protein expression, purification Methods and western blot Plant materials and growth conditions The coding region of ZmGPDH1 with the NcoI and XhoI The maize inbred line accession He-344 (provided by Hei- sites was amplified by PCR and then inserted into the longjiang Academy of Agricultural Sciences, Harbin, China) pET32a (+) vector containing 6 × His tag. The was used as the plant material in this experiment and nor- pET32a-ZmGPDH1 plasmid was transformed into the mally planted in a growth chamber under controlled photo- Escherichia coli (E. coli) Rosetta strain and the expres- period and temperature (12 h light/12 h dark, 23 ± 2 °C), sion of ZmGPDH1 was induced with 1 mM IPTG to with a photon flux density of 1000 μmol m− 2 s− 1. generate the putative recombinants. Then the The seeds of T-DNA insertion mutants of AtGPDHc2 His-tagged ZmGPDH1 proteins were extracted and puri- (TAIR: At3G07690), namely atgpdhc2 (SALK_033040) fied under native conditions using Ni-NTA nickel col- were donated by Dr. Pradeep Kachroo (University of umns (Sigma), and the purified proteins were detected Kentucky, USA). The homozygous lines of atgpdhc2 mu- by 12% SDS-PAGE as well as Western blot using 6 × His tant were identified by PCR and reverse transcription Tag Antibody as probe. PCR (RT-PCR) analysis, and the primers were shown in Additional file 6: Table S1. The plants of Arabidopsis Phenotypic analyses of the ZmGPDH1 transgenic thaliana (ecotype Col) and atgpdhc2 mutant (ecotype Arabidopsis under salt or osmotic treatment Col) were grown in a growth chamber under controlled For germination analysis, seeds of WT, atgpdhc2, OE photoperiod and temperature (16 h light/8 h dark, 21 ± and COM lines were plated on half-strength MS plates 2 °C), with a photon flux density of 100 μmol m− 2 s− 1. containing 200 mM mannitol or 100 mM NaCl for 8 days. Germination rates were counted at day 5 after Plasmid construction and plant transformation sowing and seed germination was defined as the appear- The full length coding region of ZmGPDH1 (1377 bp) was ance of visible radicle. To investigate the effects of salin- cloned from cultivated maize by the gene specific primers ity and osmotic stresses on root length and fresh weight, (Additional file 6: Table S1). The PCR product was puri- 7-days-old seedlings of WT, atgpdhc2 mutant, COM and fied and inserted into the XbaI and SalI sites of the OE lines were transferred into half-strength MS plates pBI121-GFP vector under control of the CaMV35S pro- supplemented with 150 mM NaCl or 300 mM mannitol. moter. For complementation and over-expression assays, The root length and fresh weights of stress-treated seed- Agrobacterium tumefaciens strain EHA105 carrying the lings were determined after 7 day of treatment. construct pBI121-ZmGPDH1::GFP was used to transform For the stress tolerance test at the adult stage, Arabidopsis wild-type or atgpdhc2. T3 homozygous trans- 3-week-old Arabidopsis plants were irrigated with 200 genic Arabidopsis were screened by RT-PCR. mM NaCl or 400 mM mannitol solution every 3 days for a total of 9 days. Rosette leaf samples were collected at Protein subcellular localization and GUS activity assay day 6 of treatments to measure the changes of various To verify the subcellular localization of ZmGPDH1, the physiological and biochemical parameters. All experi- mesophyll protoplasts were isolated from T3 homozygous ments were replicated at least three times with 80–100 transgenic Arabidopsis harboring pBI121-ZmGPDH1::GFP plants per treatment. Photographs taken from one repre- or pBI121-GFP (control) plasmid, and the subcellular sentative experiment are shown. Total chlorophyll localization of GFP expression was visualized by confocal (chlorophyll a + b) was determined according to the laser-scanning microscope (Leica, German). The positive method as previously described [42] and the fresh young control (empty vector) or fusion proteins were also tem- leaf was extracted in 80% (v/v) acetone extract. Photo- porarily expressed in rice mesophyll protoplasts according chemical efficiency (Fv/Fm) was examined by using a pul- to the methods described previously [40]. For se-modulated fluorometer (FMS2, Hansatech, UK) [43]. co-localization studies, a far-red fluorescent protein mkate To investigate the water loss rate (WLR), the rosette was used as the cytosol marker. To study the promoter ac- leaves from 4-week-old Arabidopsis were weighed at tivity, a 1642-bp genomic region upstream of the transla- specific time points. The decrease in fresh weight was tion initiation codon of ZmGPDH1 gene was cloned into used to calculate WLR. For stomatal closure assays, the pBI121-GUS at HindIII and XbaI sites (primers see Add- strips abaxial epidermis of Arabidopsis leaves were im- itional file 1: Table S1). The constitutive proZmGPDH1::- merged in buffer (10 mM MES/KOH, pH 6.1, 10 mM GUS transformed WT plants were also generated and T3 KCl, 50 μM CaCl2) under light for 1 h to induce the sto- homozygous transgenic lines were used for GUS staining matal opening and then treated with 300 mM mannitol, Zhao et al. BMC Plant Biology (2019) 19:16 Page 15 of 17 150 mM NaCl and 20 μM ABA for 3 h. The conform- Quantitative real-time RT-PCR analysis ation of stomatal aperture were photographed by con- To analyze the expression of ZmGPDH1 under osmotic focal microscope and processed with ImageJ software. and salt stresses, 3-week-old maize seedlings were treated The experiments were replicated at least three times with 1/2 Hoagland solution containing 400 mM mannitol with 40–50 cells per treatment. and 200 mM NaCl solutions for 0, 1, 3, 6, 12 and 24 h, and the roots were sampled to analyze the transcripts of ZmGPDH1. The untreated maize samples from the same Analysis of GPDH activity, G-3-P and glycerol levels time point were used as the controls. ZmGAPDH and G-3-P and glycerol contents were measured as previ- ZmACTIN genes served as internal reference in each ously described with slight modifications [44]. The assay. To examine the tissue-specific expression of GPDH activity was examined with regard to the reduc- ZmGPDH1, total RNA was extracted from rosette leaves tion of DHAP by NADH. The total reaction volume of (RL), flower buds (FLB), roots (RT), flowers (FL), siliques the assay was 1 mL containing 100 mM HEPES buffer, (SL), stems (ST) and cauline leaves (CL) in pH 6.9, 4 mM DHAP, 0.2 mM NADH and an appropriate proZmGPDH1::GUS transgenic plants. To analyze target amount of enzyme [8]. The absorbance changes at 340 genes expression induced by osmotic and salt stresses, nm were monitored using an ultraviolet spectrophotom- 3-week-old WT, atgpdhc2, and OE lines were treated with eter (U3900, Hitachi High-Technologies, Japan). water (control), 300 mM mannitol or 150 mM NaCl solu- tion. Total RNA was extracted from rosette leaf samples Analysis of cellular redox and ROS homeostasis at 24 h after treatments. The expression of target genes in The reduced pyridine nucleotides (NADH) content, WT plants under control environment was used as a cali- oxidized pyridine nucleotides (NAD+) content and brator. ACTIN2 and UBQ7 genes were used as internal NADH/NAD+ ratio were assayed with an enzymatic reference [56]. The primers used for transcriptional ana- cycling procedure [45]. The ascorbate (ASA) content, lysis were shown in Additional file 6: Table S1. dehydroascorbate (DHA) content and ASA/DHA ratio were measured following the reported protocols [46]. Statistical analysis The glutathione (GSH) content, oxidized glutathione Data are presented as Mean ± SD. The Student’s t-test (GSSG) content and GSH/GSSG ratio were assayed as was used to determine the significance levels using SPSS described [47]. 21.0 software throughout this study. A P-value of < 0.05 For ROS accumulation analysis, the staining of was considered statistically significant. nitroblue tetrazolium (NBT) and 3,3- diaminobenzi- dine (DAB) of stress-treated seedlings were performed following the reported protocol [48]. For hydrogen Additional files peroxide (H2O2) staining in the guard cells, prepared Additional file 1: Figure S1. Alignment analysis of the ZmGPDH1 and epidermal peels with NaCl, mannitol or ABA treat- AtGPDHc2 protein sequence (TIF 911 kb) ment were stained with 2,7-dichlorofluorescin diace- Additional file 2: Figure S2. Subcellular localization of pBI121- tate (H2DCF-DA) for 10 min [49]. Cell death caused ZmGPDH1::GFP fusion proteins in rice mesophyll protoplasts. a Confocal by salt or osmotic stress was also estimated by Evan′s micrographs showing localization of GFP and ZmGPDH1-GFP. b Confocal micrographs showing localization of ZmGPDH1-GFP in mesophyll blue and PI staining as described [24]. The assays of protoplasts expressing a far-red fluorescent protein mkate (TIF 2244 kb) H2O2 and superoxide (O2.-) were conducted by spec- Additional file 3: Figure S3. Kinetic analysis of the GPDH activity of trophotometry as previously described [50, 51]. The ZmGPDH1. (TIF 43 kb) lipid peroxidation was measured with reference to the Additional file 4: Figure S4. Molecular characterization of the atgpdhc2 thiobarbituric acid-reactive substances (TBARS) con- mutant. a Genomic organization of the atgpdhc2 location. b Identification of homozygous mutants. M: DL2000 marker; LP and RP: Forward and tent [52]. Electrolyte leakage (EL) was assessed as de- reverse primers of target genes; LB: The T-DNA left border primer. scribed [53]. c Reverse transcription PCR (RT-PCR) of AtGPDHc2 transcripts in atgpdhc2 To monitor the antioxidant enzyme activities, leaf mutants and wild-type (WT) Arabidopsis. (TIF 1762 kb) tissues (0.5 g) were ground in ice bath with 10 mL ex- Additional file 5: Figure S5. Phenotype of ZmGPDH1 OE lines in response to ABA. a The seeds of WT and OE lines were germinated on traction buffer (K2HPO4-KH2PO4, pH 7.0, 1.5 mM half-strength MS plates with or without ABA. b Germination rate of WT EDTA, 1% PVP, 0.5 mM ASC), and then the hom- and OE lines under different concentrations of ABA treatment at day 5 ogenate was centrifuged at 12000 rpm for 20 min at 4 after imbibitions. c 7-day-old WT and OE seedlings were grown on half-strength MS plates without or with ABA for 7 days. d The fresh weigh °C. The supernatant was used for the determination and primary root length of WT and OE seedlings after ABA treatment. of enzymes activities. The activities of catalase (CAT), Asterisks indicate significant differences from WT plants by Student′s t-test ascorbate peroxidase (APX) and superoxide dismutase (*P < 0.05; **P < 0.01). (TIF 10675 kb) (SOD) were determined as described [54, 55], with Additional file 6: Table S1. The gene ID and primers used in this study. (PDF 86 kb) slight modifications. Zhao et al. BMC Plant Biology (2019) 19:16 Page 16 of 17 Abbreviations Author details 1 ABA: Abscisic acid; APX: Ascorbate peroxidase; ASA: Ascorbate; CAT: Catalase; Key Lab of Modern Agric